Interactions between serine proteases, their substrates, and their inhibitors have largely been exploited during evolution. Protease cascades are not confined to the classical blood coagulation or complement cascade. Thrombin, in addition to catalyzing fibrin polymerization, can act as a novel ligand for the recently identified thrombin receptor. Vu et al., Cell 64:1057-1066 (1991). The receptor is a member of the seven-transmembrane domain receptor family that possibly mediates other known effects of thrombin, including its role as mitogen for lymphocytes and fibroblasts. Chen et al., Proc. Acad. Natl. Sci. USA 72:131-135 (1975); Chen et al., Exp. Cell. Res. 101:41-46 (1976). Hepatocyte growth factor (also known as scatter factor), which promotes cell division and epithelial morphogenesis, is similar in structure to serine proteases, having a 38% amino acid sequence identity with plasminogen, although it lacks proteolytic activity as a result of mutation of two residues in the catalytic triad. Rubin et al., Proc. Natl. Acad. Sci. USA 88:415-419 (1991); Montesano et al., Cell 67:901-908 (1991); Gherardi et al., Nature (London) 346:228 (1990); Nakamura et al., Nature (London) 342:440-443 (1989). Hepatocyte growth factor is the ligand for the c-met proto-oncogene product, a transmembrane 190-kDa heterodimer with tyrosine kinase activity that is widely expressed in normal epithelial tissues. Bottaro et al., Science 251: 802-804 (1991); Naldini et al., Oncogene 6:501-504 (1991); Di Renzo et al., Oncogene 6:1997-2003 (1991).
In analog to this finely dissected developmental system, a considerable body of evidence has pointed to a set of different proteases as prime candidates in the regulation of tumor invasion and angiogenesis. Liotta et al., Cell 64:327-336 (1991); Mignatti et al., Physiol. Rev. 73: 1-36 (1993) . The activities of these proteases are strictly regulated at the levels of both gene expression and zymogen activation. Matrisian, BioEssays 7:455-463 (1992). Moreover, the activities of most of these proteases appear to be enhanced when the enzymes are cell membrane associated. Cell-bound proteases are subject to negative regulation by natural protease inhibitors. Chen, Curr. Opin. Cell. Biol. 4:802-809 (1992). Although current knowledge of protease cascades relates to tissue remodeling during tumor invasion and angiogenesis, it is likely that other cells perform similar functions. In fact, normal tissue homeostasis is dependent on balanced rates of cell division, extracellular matrix (ECM) synthesis, and degradation. Recent evidence has demonstrated a close link between cytokines and growth factors that directly modulate these three processes. It is known that the ECM acts as a reservoir for several growth factors and modulates their activities. Flaumenhaft et al., Curr. Opin. Cell Biol. 3:817-823 (1991). There is also evidence that a number of proteases are involved in growth factor mobilization from the ECM. Barr, Cell 66:1-3 (1991); Flaumenhaft et al., J. Cell Biol. 118:901-909 (1992).
To dissect the mechanism that controls growth arrest in mammalian cells, a set of six growth arrest-specific (gas) genes: which are highly expressed during serum starvation in NIH 3T3 mouse fibroblasts have been cloned. Two of these genes, referred to as gas1 and gas2, were investigated in detail for their kinetics of induction after serum starvation and density dependent inhibition. Schneider et al., Cell 54:787-793 (1988).
More recently, a third gene, gas6, has been described in detail. Manfioletti et al., Mol. and Cell. Biol. 13:4976-4985 (1993). The gas6 gene encodes a protein which has sequence similarity with protein S, a serum protein that functions as a cofactor in a protease cascade that regulates coagulation. Tissue expression analysis of gas6 suggests that the gas6 protein is more likely to function in tissues than to play a role in serum processes. The association of gas6 expression with growth arrest suggest a possible role of the gas6 protein in the regulation of the growth of cells and tissues. The mechanism of this regulation and the precise cellular processes that gas6 participates in are not defined by these studies.
In an independent effort to identify genes that control the proliferation state of cells, a research program designed to identify novel growth factors was initiated. The strategy employed was to identify receptors for which a ligand had yet to be identified ("orphan receptors"). The orphan receptor chosen was axl, a gene involved in myeloid cell proliferation. Axl was identified by two independent laboratories as a transforming gene from cells of a patient with chronic myelogenous leukemia (O'Bryan et al., Mol. and Cell. Biol., October, 1991, pages 5016-5031), or from cells of a patient with chronic myeoloproliferative disorder (Janssen et al., Oncogene 6: 2113-2120) . Both laboratories identified axl due to its ability to render 3T3 (mouse fibroblast) cells tumorigenic. Thus, axl expression appears to have profound effects on the growth state of cells. Molecular analysis of the cloned axl cDNA revealed that the axl gene coded for a novel receptor tyrosine kinase. The association of axl expression with myeloid malignancies and the ability of axl to transform cells suggest that axl functions in regulating the growth status of cells. Normally, the activity of a receptor is regulated by ligand binding. Therefore, the ligand for axl may regulate growth of cells and tissues that express the receptor. Tissues that express axl are known to include bone marrow, thymus, spleen, ovary, bladder, heart and brain.